AU604534B2

AU604534B2 - A combination of cholecalciferol derivatives for the treatment of renal bone disease

Info

Publication number: AU604534B2
Application number: AU12747/88A
Authority: AU
Inventors: Shmuel Edelstein; John Anthony Kanis; David Ladkani; Zeev Mazor; Mordecai Popovtzer; Benjamin Shalita; Ben Zion Weiner
Original assignee: Teva Pharmaceutical Industries Ltd
Current assignee: Teva Pharmaceutical Industries Ltd
Priority date: 1987-07-22
Filing date: 1988-03-07
Publication date: 1990-12-20
Anticipated expiration: 2008-03-07
Also published as: JPS6431722A; DE3873855D1; EP0301167B1; DE3873855T2; ZA881307B; US4997824A; AU1274788A; EP0301167A1

Description

I.'

04.0 IN M3 2.0 ~L8 06BL99PEZ LzAX-A~sjbdouwP11!!4~j ap~q0 ZAXMAnisedONrIN IH EJ 0)V 'Id 8 068L9VCZ ZAXMAn~s.ibdouW1J!iL(31p:)q Z~kXMAnisdodNNw~rIH0rnQD9V 'Id 01 54,0

U

1 2 5 I11b11.

_1.25 .4 I6 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 SUBSTITUTE COMPLETE SPECIFICATION FOR OFFITCE USE Form Short Title: Int. Cl; Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: 00 009 000 000 00 0000 0 0 .00.0 000000 0 Priority: This document contains the amendments made uindLr- Section 49 and is correct for printing.

Related Art: TO BE COMPLETED BY APPLICANT 0 0 00 0 0 00 0 Name of Applic.ant: Address of Applicant: Actual Inventor: Address for Service: TEVA PHARMACEUTICAL INDUSTRIES LTD.

Jerusalem, ISRAEL Mordecai Popovtzer; Ben Zion Weiner; Shmuel Edelstein; Zeev Mazor; David Ladkani; Benjamin Shalita and John Anthony Kanis GRIFFITH HASSEL FRAZER 71 YORK STREET SYDNEY NSW 2000

AUSTRALIA

Complete Specification for the invention entitled: A COMBINATION OF CHOLECALCIFEROL DERIVATIVES FOR THE TREATMENT OF RENAL BONE DISEASE The following statement is a full description of this in,-.ntion, including the best method of performing it known to me/us:- 3136A:rk TO: THE COMMISSIONER OF PATENTS 7 7R I COMMONWEALTH OF AUSTRALIA 0306A:rk S (,i PKICATION ACCEPTED AND AMENDMENTS Si

-?I

The invention relates to an improved method and pharmaceutical composition for the treatment of human bone diseases. More particularly the invention provides a method and composition for the treatment of renal osteodystrophy diseases in humans comprising the administration to a patient of 6-40 micrograms per day of 24,25 dihydroxycholecalciferol (hereafter "24,25(OH) 2

D

3 in combination with l-alpha-hydroxycholecalciferol (hereafter "1-alpha(OH)D 3 or 1,25-dihydroxycholecalciferol (hereafter "1-25(OH) 2

D

3 or dihydrotachysterol (hereafter "DHT2") in an amount per day which will maintain the patient's calcium serum concentration at 10-11 mg/dl and the phosphate concentration at 5.5-6.0 mg/dl.

As used herein, means "day" and "dl" means "deciliter". Also "mcg" means "micrograms" and "mg" means "milligrams".

S

o BACKGROUND OF THE INVENTION Cholecalciferol (vitamin D 3 has been known to be intimately associated with bone metabolism and effectively S used for the cure of rickets and osteomalacia since the early 1920s. Studies carried out during the last decade have shown that vitamin D 3 must be metabolically activated 04s before functioning biochemically in its target tissues, .?25 including the intestine, bone and kidney. The o dihydroxylated metabolite of vitamin D 3 namely 1,25(OH) 2

D

3 was believed to be responsible for all the 2 V com-Pund known biological functions of the vitamin. This4-eempui-d-is o produced from cholecalciferol by C-25 hydroxylation in the liver followed by C-1 hydroxylation in the kidney. It was thus understandable why treatment with cholecalciferol was found to be ineffective in metabolic bone diseases, such as renal osteodystrophy, in patients who, owing to chronic kidney failure, are incapable of converting the cholecalciferol prohormone into its aforesaid dihydroxylated metabolite hormone.

6S:ND 2 NT GRIFFITH HI-ASSEL FRAZER, SYDNEY, AUSTRALIA 3706A:rk 1 The use of 1,25(OH)2D 3 is now established in the treatment of renal bone diseases. Its administration increases calcium absorption from the gut and consequently, plasma calcium, and suppresses secondary hyperparathyroidism and its skeletal consequences. It also ameliorates osteomalacia in the presence of secondary hyperparathyroidism. The putative roles of 1,25(OH) 2

D

3 in the bone are controversial and many of its actions can be accounted for by its effect to increase the ionized fraction of plasma calcium. This therapeutic effect is also the major cause of vitamin D toxicity, namely hypercalcemia.

Its use is therefore contraindicated and indeed of limited value in patients with pre-existing hypercalcemia due to aluminum toxicity or tertiary hyperparathyroidism.

The failure of 1,25(OH) 2

D

3 to control secondary oLo hyperparathyroidism in many patients on dialysis stimulated the continuing search for more effective therapeutic means.

Intravenous administration of 1,25(OH) 2

D

3 has been recently reported to be more effective than the same drug administered by the oral route in suppressing secondary S hyperparathyroidism (Slatopolsky et al., J. Clin. Invest 74, 2136, 1984). However, it has not solved the problem of secondary hyperparathyroidism in dialysis patients.

o l-alpha(OH)D 3 is a synthetic analog of o >3 1,25(OH) 2

D

3 It is converted into the latter in the S liver by 25-hydroxylation. l-alpha(OH)D 3 is now also in o"L clinical use for renal osteodystrophy, and its therapeutic effect is equivalent to that of 1,25(OH) 2

D

3 apart from small differences in its biological half-life and dose ":SO response.

Although the treatment of chronic renal failure (CRF) with either 1,25(OH) 2

D

3 or l-alpha(OH)D 3 proved to be effective in maintaining normal concentrations of calcium and phosphate in the plasma, the beneficial results on the mineralization of bone matrix were highly incomplete and unsatisfactory.

5996S:ND 3 There has been recent interest in other metabolites of vitamin D 3 notably 25(OH)D 3 and 24,25(OH) 2

D

3 which may have different actions on target tissue than 1,25(OH) 2

D

3 and l-alpha(OH)D 3 Until recently it was still controversial whether 24,25(OH) 2

D

3 plays any physiological role in man and in animals, and whether it possesses a defined biological activity. In early observations this metabolite was considered to be only a by-product of renal 25(OH)D 3 metabolism. The uncertainty surrounding the physiological importance of 24,25(OH) 2

D

3 stems partially from observations in animals.

In animal experiments, the effect of 24,25(OH) 2

D

3 on stimulation of intestinal calcium absorption was contingent upon prior renal hydroxylation to c, 1,24,25(OH)3D 3 Thus in nephrectomized animals 24,25(OH) 2

D

3 failed to stimulate intestinal transport.

Conversely in man where 24,25(OH) 2

D

3 has been shown to be active in calcium metabolism 1,24,25(OH) 3

D

3 was found S20 to exert only a minimal effect. (Boyle et al., J. Biol.

t Chem. 248, 4174, 1973).

The increased calcium retention of 24,25(OH) 2

D

3 is not associated with hypercalkemia, suggesting that 24,25(OH) 2

D

3 may be directly or indirectly promoting calcium uptake in compartments other than the extracellular fluid. There is some evidence that skeletal retention of Scalcium is augmented. A number of experimental observations S in animals suggest that both 1,25(OH) 2

D

3 and 24,25(OH) 2

D

3 are necessary for some aspects of skeletal metabolism. These include the normal differentiation of cartilage tissue, the healing of fractures and the I t mineralization of rachitic bone and cartilage and the prevention of parathyroid gland hyperplasia.

5996S:ND 4 RENAL OSTEODYSTROPHY Renal bone disease is a common complication in patients undergoing chronic dialysis.

Osteitis fibrosa cystica and osteomalacia are the two most common forms of uremic osteodystrophy. The typical histological features of osteitis fibrosa cystica are osteoclastic bone resorption forming cystic defects, endosteal and marrow space fibrosis with aggregates of osteoclasts and brown tumors. In addition there are morphological indications of rapid bone turnover reflected by increased active osteoid surface, increased formation of woven bone and enhanced tetracycline uptake. The characteristic roentgenographic features are subperiosteal bone resorption, cystic defects, frequently associated with osteoclerosis and extraskeletal calcifications. Secondary hyperparathyroidism that is responsible for osteitis fibrosa cystica, emerges early in the course of chronic renal fill disease. (Popovtzer et al., Clin. Sci. 38 297, 1970).

In chronic renal disease, loss of filtering units imposes increasing loads of phosphate on the remaining i nephrons. The increased burden of phosphate may have seVera e-r-e-r-a--secondary consequences. The high phosphate in serum level causes a reciprocal fall in serum ionized calcium and the latter triggers secretion of parathyroid hormone (PTH). PTH restores both the serum phosphate and calcium to normal through its phosphaturic and calcemic S actions. Subsequent reductions in nephron population will evoke a further rise in PTH leading to secondary 1 hperparathyroidism as an end-result (Popovtzer-ot al. Inter.

J. Artif. Org., 3, 1, 1980).

Reduced 1,25(OH) 2

D

3 activity, as demonstrated to be I present in early renal disease, may cause secondary severcL hyperparathyroidism by-se-rve-a- mechanisms, reduced intestinal absorption of calcium, diminished skeletal calcemic response to PTH, and loss of physiological suppression of PTH secretion.

S6:ND 5 o 1 Patients with renal bone diseases are often divided into two subgroups, an active bone disease subgroup and a non-active bone disease subgroup. This is done in accordance with the severity of the bone disease, particularly with regard to osteoclastic activity and bone forming activity, reflected by active osteoid and tetracycline uptake levels.

Often, in active bone disease, the osteoclast number equals or exceeds 0.3 mm-land the active bone resorption equals or exceeds Patients who do not meet these two criteria are included in the non-active bone disease subgroup.

Description of Prior Art Ornoy et al (Nature, 276, 517, 1978) demonstrated that S, the combined administration of l-alpha(OH)D 3 with 24,25(OH) 2

D

3 produced a better cure of mineralization defect than l-alpha(OH)D 3 alone in vitamin D deficient chicks. Galus et al (Calcif. Tissue Int., 31, 209, 1980) have shown that the administration of 24,25(OH) 2

D

3 in S vitamin D-deficient dogs increases bone formation and 4 mineralization and decreased bone resorption, while l-alpha(OH)D 3 increased both mineralization and resorption. Pavlovitch et al Clin. Invest., 68, 803, 1981) demonstrated an inhibition of increased bone S' resorption after acute bilateral nephrectomy by 24,25(OH) 2

D

3 in rats. In addition 24,25(OH) 2

D

3 S blocked partially the stimulating effect of 1,25(OH) 2

D

3 on bone resorption. Rubinger et al (Proc. 6th Workshop Vit.

D, Italy, 1985, p. 96) demonstrated that 24,25(OH) 2

D

3 suppressed the hypercalcemic effect of 1,25(OH) 2

D

3 in rats with chronic renal failure.

5996S:ND 6

I

Tests in Human Patients Kanis et al. (Brit. Med. J. 1382, 1978) suggested that 24,25(OH) 2

D

3 may be an important regulator of skeletal metabolism in man with potential value as a therapeutic agent in patients with chronic renal failure and osteoporosis.

Weisman et al (Brit. Med. J. 281, 712, 1980) indicated generally that treatment with both 1,25(OH) 2

D

3 and 24,25(OH) 2

D

3 may be needed to prevent and heal renal osteodystrophy.

In Japanese Patent Application No. 55 139-320, published on October 31, 1980, cominations of 24,25(OH) 2

D

3 with either 1,25(OH) 2

D

3 or l-alpha(OH)D 3 are claimed for the treatment of numerous bone and calcium metabolic diseases, amongst them also renal o 0 bone dystrophia. However, this patent application is very So general and vague and fails to disclose how to use these 000 oo8o combinations in any of the diseases mentioned, including oo renal osteodystrophy.

0 20 Dunstan et al. (Mineral Electrolyte Metab. 11, 358, o 1985) concluded that "1,25(OH) 2

D

3 is a useful agent in oo ooE the treatment of renal bone diseases but no therapeutic value is apparent for 24,25(OH) 2

D

3 Thus, the above publication teaches away from a combination of 24,25(OH) 2

D

3 with 1,25(OH) 2

D

3 and 0oo consequently also with l-alpha(OH)D 3 which is 0 00 o00o' biochemically and therapeutically equivalent to 0 1,25(OH) 2

D

3 o00 0 SUMMARY OF INVENTION SThe invention provides a novel method and 0000 ooo pharmaceutical composition for the improved treatment of human renal osteodystrophy patients undergoing hemodialysis.

According to one aspect of the invention, the new method comprises the administration to such a patient 6-40 mcg/d of 24,25(OH) 2

D

3 in combination with daily amounts of l-alpha(OH) D 3 or 1,25(OH) 2

D

3 or DHT 2 which will 5996S:ND 7 i x;I suffice to maintain normal calcium levels, namely at 10-11 mg/dl, and the phosphate concentration at 5.5 6.0 mg/dl, and that the numerical value of the multiplication of the calcium and the phosphate concentrations in the serum should be about 60-65.

The ingredients in the combination may be administered separately, all of them orally by means of capsules or, if preferred, l-alpha(OH)D 3 and 1,25(OH) 2

D

3 parenterally.

The preferred method of administration is orally by means of capsules containing 5 mcg of 24,25(OH)2D 3 and various amounts of l-alpha(OH)D 3 or 1.25(OH) 2

D

3 such as 0.25 mcg, 0.5 mcg, 0.75 mcg and 1.0 mcg per capsule.

The new method of treatment of chronic renal failure (CRF) patients by means of the specific combinations of 24,25(OH) 2

D

3 with 1-alpha(OH)D 3 or 1,25(OH) 2

D

3 in the relative amounts of each component as stated above o offers significant improvements in the clinical symptoms, in ooo the various bone histological parameters and blood biochemistry of these patients.

O 20 Another aspect of the invention is the use of the novel 0 o combination of 24,25(OH) 2

D

3 with l-alpha(OH)D 3 or 0 O 1,25(OH) 2

D

3 in the stated relative proportions for CRF patients simultaneously with other drugs which may be used in their treatment. In addition dihvdrotachysterol 025 (DHT 2 which has been shown to be effective in combination with 24,25(OH) 2

D

3 in the treatment of renal "o 0 osteodystrophy in adults and children can be used in 0 0 combination with 24,25(OH) 2

D

3 (van Diemen-Steenvoorde, 0 a et al., Clin. Nephrol., 24, 292, 1985).

DETAILED DESCRIPTION OF INVENTION 0 The clinical treatment at present in practice for patients with renal bone diseases, including those undergoing hemodialysis, comprises the administration of either 1,25(OH) 2

D

3 or l-alpha(OH)D 3 or DHT 2 orally or parenterally.

5996S:ND 8 L I 1 ~L~I' l~i l~ r- Although these cholecalciferol analogs proved to be effective in maintaining normal concentrations of calcium and phosphate in the plasma of the patients, they showed little beneficial results on the bone mineralization of the bone matrix. The treatment consequently failed to improve clinical bone symptoms and bone histological parameters of the patients suffering from osteitis fibrosa cystica resulting from secondary hyperparathyroidism and from osteomalacia. Although some Prior Art publications indicated that both 1,25(OH) and 24,25(OH) 2

D

3 may be necessary for skeletal metabolism and bone formation and mineralization, and other publications suggested that the combined treatment of the above two cholecalciferol metabolites in renal osteodystrophy may have some therapeutic advantages, there was no disclosure in the Prior Art of how to perform or use such a combination which will result in any advantageous therapeutic effects.

By virtue of the present invention there was discovered a specific method and pharmaceutical combination of S 20 24,25(OH) 2

D

3 with either l-alpha(OH)D, 1,25(OH) 2

D

3 or DHT 2 which will offer considerable therapeutic advantages to renal osteodystrophy patients (also called chronic renal failure CRF patients).

Studies have been provided where the patients were 25 randomly allocated into two groups. One group of patients was treated with 1-alpha(OH)D 3 alone or 1,25(OH) 2

D

3 alone (hereafter the "control group") in daily dosages to maintain normal serum concentrations of calcium and phosphate. To the second group of patients (hereafter the "treatment group") was administered 24,25(OH) 2

D

3 in daily dosages ranging from 10 mcg to 40 mcg in combination with l-alpha(OH)D 3 or 1,25(OH) 2

D

3 in daily amounts which would maintain the patients calcium(Ca) serum concentrations at 10-11 mg/dl and phospate serum concentration at 5.5-6.0 mg/dl, and the Ca x P numerical product should not exceed 60-65.

5996S:ND 9 L_ The daily dosages of l-alpha(OH)D 3 or 1,25(OH) 2

D

3 in both the "treatment group" and in the "control group" were adjusted for every patient individually, in order to maintain the serum concentrations of calcium and phosphate at the normal range levels as stated above.

The actual daily doses of l-alpha(OH)D 3 or 1,25(OH) 2

D

3 administered to the patients ranged from 0.25 mcg to 3.0 mcg.

The controlled studies have surprisingly shown that significant therapeutic improvements were achieved in the treatment group in comparison with the control group.

Improvements were observed in both aspects of the renal bone disease, in the bone histological parameters as well as in oo the relief of the bone clinical symptoms.

0 0 o °ooo Bone Histomorphometrv 0000 oo Two bone biopsies were obtained from the iliac crest, 0 C, one at the outset of the study and the other upon its

OO

0 completion. The histomorphometry of the biopsies showed the .0 following favourable results: The trabecular bone volume in the treatment group vras considerably lower than in the control group.

The active osteoid surface was significantly reduced in oo°° the treatment group compared to the control group. This was shown in the second biopsy, especially in patients with 0 relatively high active osteoid surface levels in the first o 0o biopsy.

In the resorption surface, a statistically significant o decrease was observed in the treatment group, whereas no change could be detected in the control group.

The improvements in the bone resorption were even more pronounced in the active bone disease subgroup, which was defined as having an osteoclast number equal to or exceeding 1- 0.3 mm and active bone resorption equal to or exceeding All patients with active bone disease in the treatment group showed a significant decrease in bone 5996S:ND 10 resorption, whereas in the control group there was no change or even an increase in bone resorption.

Clinical symptoms With respect to clinical manifestations before and after treatment in the active bone disease subgroup, such as bone pains and tenderness, muscle pains and pruritus, there was a significant improvement in the clinical symptoms in the treatment group. In the control group, the clinical symptoms remained unchanged, or even worsened.

Untoward Side Effects Careful observation in all studies have not revealed o0 untoward side effects in the treatment group that could be °5 attributed to 24,25(OH) 2

D

3 given orally at a daily dose eat of either 10 mcg or 40 mcg over the whole period of the o 0000 0o studies. Thus 24,25(OH) 2

D

3 can be considered a "very S" safe drug" when administered at the abovementioned daily ooa dosage. In addition, it was found that 24,25(OH) 2

D

3 ?0 administered in combination with l-alpha-hydroxylated D 3 000000 0 derivatives increased the patient's tolerance of these derivatives and reduced the risk of hypercalcemia.

o"o. The best mode of carryina out the invention 0 00 0 oO25 The clinical trials and studies of the present a 0 00 S0 invention indicate that the best mode of carrying out the 0 invention is as follows: 000 The treatment of patients with renal osteodystrophy, including those undergoing chronic hemodialysis, should °0030 comprise the administration to each patient of 10 mcg per 000 day of 24,25(OH) 2

D

3 in combination with either 1-alpha(OH)D 3 1,25(OH) 2

D

3 or DHT 2 in daily doses which will maintain the patient's calcium concentration at the upper normal levels of 10 to 11 mg/dl and the phosphate serum concentration at 5.5-6.0 mg/dl. Another criterion for the daily dosage of l-alpha(OH)D 3 or DHT 2 should be that the numerical value of the multiplication "calcium x phosphate" should not exceed 60-65 during the treatment.

5996S:ND 11 ^ll i With the specific combination of 10 mcg per day of 24,25(OH) 2

D

3 and the appropriate amounts of l-alpha(OH)D 3 1,25(OH) 2

D

3 or DHT 2 the most favorable therapeutic effects were obtained in the treatment of renal osteodystrophy patients considering all the aspects of the disease: The blood biochemical and hematological measurements, the bone histomorphometry and the clinical manifestations. These favorable results are outlined in detail in example 7 (Study I).

The rationale for the administration of 10 mcg of 24,25(OH) 2

D

3 per day to patients can be based on calculations obtained from the physiological and therapeutic experiments conducted in the chick. The optimal oo physiological dose of 24,25(OH) 2

D

3 in the chick was 0 2 3 found to be 0.3 mcg, which is about 3 times that of °o 1,25(OH) 2

D

3 or 1-alpha(OH)D 3 As the therapeutic 0000ooo oo daily doses of the latter in the human are 20 times the physiological doses, the daily therapeutic dose of 0° 24,25(OH) 2

D

3 in the human should be at least 20 x 0.3 20 mcg, namely 6 mcg.

000000 a0 The best daily dose of 24,25(OH) 2

D

3 in CRF patients was found by the present invention to be 10 mcg which is only in slight excess of the theoretical dose of 6.0 mcg/d.

ooo0 24,25(OH) 2

D

3 should best be administered by means o oo 2 3 o oo25 of 2 soft gelatine capsules per day, containing 5 mcg per capsule, one in the morning and one in the evening.

"0 Even more preferable is the administration by means of 000 0 soft gelatine capsules containing a combination of 5 mcg 0 24,25(OH) 2

D

3 and amounts of 0.25 or 1.0 mcg of 1-alpha °°030 (OH)2D 3 or 1,25(OH) 2

D

3 in accordance with the needs of each individual patient.

In the case where 1-alpha(OH)D 3 or 1,25(OH) 2

D

3 is administered separately from 24,25(OH) 2

D

3 it can be done orally by means of soft gelatine capsules or tablets containing 0.25 mcg or 1.0 mcg of the active ingredient.

Alternatively, it can also be administered parenterally by means of ampoules containing the desired amounts of the 5996S:ND 12 1-; i active ingredient and stabilized by means of sodium ascorbate and a buffer to pH of 6.4 to 7.8 (See U.S.P.

4,308,264).

The soft gelatine capsules of all the active ingredients (l-alpha(OH)D 3 1,25(OH) 2

D

3 may be prepared in arachis oil, propylene glycol, glycerin and other suitable solvents together with a stabilizer and a preservative.

Another embodiment of the invention is the use of the combination of 24,25(OH) D in the range given with dihydrotachvsterol (DHT), which is usually administered orally by means of tablets, capsules, or solutions.

The beneficial effect of the simultaneous administration of 24,25(OH) 2

D

3 and DHT 2 was demonstrated by an uncontrolled study in patients with S dialysis related osteomalacia. (Hodsman, et al., Am. J.

Med. 74, 407, 1983). A more recent study showed also the benefits of this combination in children undergoing chronic t dialysis with hyperparathyroid bone diseases, resulting in a significant decrease in the number of osteoblasts. (van I Diemen-Steenvoorde, et al., Clin. Nephrol., 24, 292, 1985).

The combination of the invention given simultaneously with DHT 2 is expected to give even more favourable results in cases of osteomalacia and renal bone disease of secondary 3 I hyperparathyroidism.

Assumed Mode of Action of The Invention In vitro studies have demonstrated the effective anabolic action of 24,25(OH) 2

D

3 in the presence of 1,25(OH) 2

D

3 and PTH. Only combined treatment of 1,25(OH) 2

D

3 24,25(OH) 2

D

3 and PTH will induce a significant increase in bone mineral deposition in cultured bones in vitro. Each one of the components operates on a ,different cellular function related to bone formation, and jointly they have synergistic effect on bone.

5996S:ND 13 SIt is well known from the literature that, while both 1,25(OH) 2

D

3 and 24,25(OH) 2

D

3 are similar in stimulating calcium absorption at the intestinal mucosal level, they differ in the nature of their action on the bone level in renal osteodystrophy patients.

While 1,25(OH) 2

D

3 in physiological doses directly stimulates bone resorption, 24,25(OH) 2

D

3 exerts an opposite action on the bone level. Thus, the advantage of f simultaneous administration of both metabolites in the proportions of the invention to renal osteodystrophy patients is in the ability of 24,25(OH) 2

D

3 to offset the effects of 1,25(OH) 2 D (or its analogs l-alpha(OH) 2

D

3 and DHT 2 with regard to the resorbing action on the bone, but on the other hand a synergistic action with respect to gut absorption, bone mineralization, and less likely, parathyroid hormone (PTH) suppression.

The following examples provide an illustration of the invention, but should not be construed as to limit the scope of the invention.

Example 1 Soft Gelatine Capsules of l-alpha(OH)D 3 Each 0.25 mcg capsule contains: S1-alpha-HYDROXYCHOLECALCIFEROL 0.275 mcg* CITRIC ACID anhydrous 0.015 mg PROPYL GALLATE 0.020 mg S VITAMIN E (dl-alpha TOCOPHEROL) 0.020 mg i ALCOHOL dehydrated 1.145 mg PEANUT OIL (ARACHIS OIL) 98.800 mg 30 Total fill weight per capsule: 100.0 mg Includes 10 per cent excess.

5996S:ND 14 The following statement is a full description of this in ntion, including the best method of performing it known to me/us:- 3136A:rk j ii 5 q: r ii

I

I..

Example 2 Soft Gelatine Capsules of 1.0 mcg 1-alpha(OH)D 3 Each 1.0 mcg capsule contains: 1-alpha-HYDROXYCHOLECALCIFEROL 1.100 m CITRIC ACID anhydrous 0.015 m PROPYL GALLATE 0.020 m VITAMIN E (dl-alpha TOCOPHEROL) 0.020 m ALCOHOL dehydrated 1.145 m PEANUT OIL (ARACHIS OIL) 98.800 m Total fill weight per capsule: 100.0 mg Includes 10 per cent excess.

Example 3 Soft Gelatine Capsules of 0.25 mcg 1,25(OH) 2

D

3 cg* g g g g g 9 3 3 3 3 ir C

C

?0

CCC,

Each 0.25 mcg capsule contains: 1,25-DIHYDROXYCHOLECALCIFEROL CITRIC ACID anhydrous PROPYL GALLATE VITAMIN E (dl-alpha TOCOPHEROL) ALCOHOL dehydrated PEANUT OIL (ARACHIS OIL) Total fill weight per capsule: 100.0 mg *Includes 10 per cent excess.

0.275 0.015 0.020 0.020 1.145 98.800 mcg* mg mg mg mg mg Ce C C C C CCC C Example 4 Soft Gelatine Capsules of 1.0 mcg of 1,25(OH) 2

D

3 Each 1.0 mcg capsule contains: 1 -30 1,25-DIHYDROXYCHOLECALCIFEROL CITRIC ACID anhydrous PROPYL GALLATE VITAMIN E (dl-alpha TOCOPHEROL) ALCOHOL dehydrated PEANUT OIL (ARACHIS OIL) Total fill weight per capsule: 100.0 mg *Includes 10 per cent excess 1.100 0.015 0.020 0.020 1.145 98.800 mcg* mg mg mg mg mg 5996S:ND 15 l6S:ND 2 L 1

I

Example Soft Gelatine Capsules of 5.0 mcg of 24,25(OH) 2

D

3 Each 5.0 mcg capsule contains: 24R,25-DIHYDROXYCHOLECALCIFEROL CITRIC ACID anhydrous PROPYL GALLATE VITAMIN E (dl-alpha TOCOPHEROL) ALCOHOL dehydrated PEANUT OIL (ARACHIS OIL) Total fill weight per capsule: 100.0 mg Includes 10 per cent excess.

Example 6 i '15 Soft Gelatine Capsules of 10.0 mcg of 5.500 mcg* 0.015 mg 0.020 mg 0.020 mg 1.145 mg 98.800 mg 24,25(OH) 2

D

3 11.000 mcg* 0.015 mg 0.020 mg 0.020 mg 1.145 mg 98.800 mg c rrt c

IPI(

If i r F r' 11 t( I 1 Each 10.0 mcg capsule contains: 24R,25-DIHYDROXYCHOLECALCIFEROL CITRIC ACID anhydrous PROPYL GALLATE VITAMIN E (dl-alpha TOCOPHEROL) ALCOHOL dehydrated PEANUT OIL (ARACHIS OIL) Total fill weight per capsule: 100.0 mg 25 *Includes 10 per cent excess.

C

"C0 CC C 5996S:ND 16 59 9 6S :ND -3 t I I Example 7 Soft Gelatine Capsules containing combination of 0.25 mcg or 1.0 rncg of 1-alpha(OH)D 3 plus 5 rncg of 24,25 (OH) 2 D 3 Each 1.0 mcg capsule contains: 1-a ipha-HYDROXYCHOLECALCIFEROL 24R, 25-DIHYDROXYCHOLECALCIFEROL CITRIC ACID anhydrous PROPYL GALLATE VITAMIN E (dl-alpha TOCOPHEROL) ALCOHOL dehydrated PEANUT OIL (ARACHIS OIL) Total fill weight per capsule: 100.0 mg i5 *Includes 10 per cent excess.

1.100 mcg* 5.500 mcg* 0.015 mg 0.020 mg 0.020 mg 1.145 mg 98.800 mg Er t ,r,

A

W

0 I 11 0 Example 8 Soft Gelatine Capsules containing combination of 0.25 mcg or 1.0 mcg of l,25(OH) 2

D

3 plus 5 mcg of 24 ,25 (OH) 2 D 3 Each 1.0 mcg capsule contains: 1,25-DIHYDROXYCHOLECALCIFEROL 1.100 mcg* 24R, 25-DIHYDROXYCHOLECALCIFEROL 5.500 rncg* CITRIC ACID anhydrous 0.015 mg PROPYL GALLATE 0.020 mg VITAMIN E (dl-alpha TOCOPHEROL) 0.020 mg ALCOHOL dehydrated 1.145 mg PEANUT OIL (ARACHIS OIL) 98.800 mg Total fill weight per capsule: 100.0 mg Includes 10 per cent excess.

5996S :ND -1 17 5996S:ND 4 I Example 9 Treatment of Chronic Renal Failure (CRF) patients with a combination of 24,25(OH) 2

D

3 and l-alpha(OH)D 3 (Study I).

The study included 56 CRF male and female adult patients undergoing chronic hemodialysis at several medical centers. The patients were randomly allocated into two groups, one group of 31 patients was treated with a combination of 24,25(OH) 2

D

3 and l-alpha(OH)D 3 (the 'treatment group") and the other group of patients was treated with l-alpha(OH)D 3 alone (the "control group").

Patients of both groups were administered 1 l-alpha(OH)D 3 in capsules of 0.25 mcg or 1.0 mcg per 1 5 capsule. The dose was adjusted for every patient individually so as to maintain the serum calcium at the upper normal level, but not higher than 11 mg/dl, and the j phosphate level not higher than 6.0 mg/dl or that the numerical multiplication product of calcium x phosphate did not exceed 65. The actual daily doses of l-alpha(OH)D 3 ranged from 0.25 mcg to 3.0 mcg.

The treatment group was administered in addition to 1-alpha(OH)D 3 also 24,25(OH) 2

D

3 at a daily dose of mcg by means of 2 capsules containing 5 mcg per capsule, one in the morning and the other in the evening.

1 The dose of 10 mcg of 24,25(OH) 2

D

3 was constant throughout the study which lasted 10-16 months.

Three major categories of variables were measured and monitored during the treatment. These were: blood biochemistry, bone histomorphometry (static and dynamic), and clinical follow-up.

I Blood Biochemistry As stated before, special attention was given to the blood levels of calcium, phosphate and to the multiplication product of calcium x phosphate.

5996S:ND 18 I S5A6S:ND 5 1M' The serum calcium concentration was found to be significantly higher in the treated group during the period 3-8 months of the study, in comparison with the control group though the levels remained within the normal range, I An increase in the 24,25(OH) 2

D

3 serum levels in the treated groups was the only significant change between the two groups during the whole study.

Bone histomorphometry One bone biopsy was taken from all patients under local anesthesia applied to both plates of the iliac crest at the onset of the study, and a second biopsy was obtained in a similar fashion from the contralateral side of the iliac jcrest approximately one year later, upon the completion of the study.

The results obtained from the biopsies showed statistically significant changes between the control group and the treatment group. These were as follows: ?0 trabecular bone volume This measurement increased significantly during treatment in the control group only.

At the end of the study it was much lower in the treatment group compared to the control group.

25 active osteoid surface The relatively high active osteoid surface levels shown by some patients at the first if biopsy were significantly lower at the second biopsy only in the treatment group and not in the control group.

30 resorption surface Generally, a statistically significant decrease in the resorption surface was shown only in the treatment group. There was no significant L change in the control group. At the end of the study, the resorption surface in the treatment group was significantly lower than that in the control group.

3D3 0; U 19 5996S:ND 6 j l Even more pronounced differences in the histomorphological results were shown in those patients in whom the respective parameters exhibited high levels at the initial biopsy, namely in patients with a more severe bone disease.

In view of this observation the patients in the study were subdivided into two types of bone disease, an active bone disease subgroup and a non-active bone disease subgroup. The division was made according to the osteoclast number and the active resorption surface.

Patients with an osteoclast number equal to or exceeding 0.3 mm-1 and with active resorption surface equal to or exceeding 2.0% were included in the active bone disease subgroup, and those with lower values were included in the non-active bone disease subgroup.

The results of this study show that all CRF patients with active bone disease in the treatment group showed a most significant decrease in active resorption surface, whereas patients with active bone disease in the control i I S0 group either showed no change or even an increase in this parameter.

However, also in the non-active bone disease subgroup 11 out of 12 patients who were treated with the combination showed a significant reduction in the active resorption surface; in contrast, the control group showed no reduction.

Clinical follow-up With respect to clinical manifestations of the disease of the treated patients, such as bone pains and tenderness, muscle pains, pruritus and others, there was generally a marked improvement only in the treatment group. This improvement was especially marked in the treatment active bone disease subgroup where most patients became asymptomatic. No relief of these clinical symptoms was observed in the control group.

No untoward side effects could be detected in the patients treated with the combination which could be attributed to 24,25(OH) 2

D

3 administered orally at a daily dose of 10 mcg.

5996S:ND 20 of 1-alpha(OH)

D

3 or 1,25(OH) 2

D

3 or DHT 2 which will 5996S:ND 7 i This study thus demonstrates that the administration of 24,25(OH) 2

D

3 to renal osteodystrophy patients in daily doses of 10 mcg in combination with 1-alpha(OH)D 3 in doses sufficient to maintain the levels of calcium and phosphate in the upper normal range, results in marked improvements in the bone parameters as well as in clinical manifestations, in comparison with the control gioup which was treated with 1-alpha(OH)D 3 alone in similar doses.

Example Treatment of CRF patients with a combination of 24,25(OH) 2

D

3 and 1-alpha(OH)D 3 (Study II) The study included 62 CRF male and female adult patients undergoing chronic hemodialysis and lasted for 10-22 months. As in Study 1, the CRF patients were randomly allocated into two groups: 34 patients were allocated to the combination treatment of 24,25(OH) 2

D

3 and 1-alpha(OH)D 3 In this study the daily dose of 24,25(OH) 2

D

3 was 40 mcg and that of l-alpha(OH)D 3 was sufficient to maintain the concentration of calcium and phosphate in the serum at the upper normal level in a manner as stated in Study I.

The patients in the control group received 1-alpha(OH)D 3 alone in similar doses as in Study I.

The blood chemistry results were similar to those obtained in Study I.

The bone histomorphometric measurements performed in patients (25 in each group) showed that the combination treatment resulted in the following favorable results: 1) a decrease in the trabecular bone volume 2) a decrease in the resorption surface 3) a slight decrease in the active resorption surface 4) a slight decrease in osteoclast number and a slight decrease in fibrotic surface.

5996S:ND 21 i i 9 I

II

These improvements in the bone parameters were not as significant as in Study I. The improvements in the clinical symptoms were also not as prominent in the treatment group in comparison with the control group as was observed in Study I.

Example 11 Combined treatment of CRF patients with 24,25(OH) 2

D

3 and 1,25(OH) 2

D

3 (Study III) IThe study included 62 CRF patients with bone diseases, such as osteitis fibrosa cystica and osteomalacia. The patients were randomly allocated into three groups.

The control group A which included 25 patients who were treated with 24,25(OH) 2

D

3 alone at doses of mcg/d.

The control group B which included 24 patients who were treated with 1,25(OH) 2

D

3 alone at varying daily doses ranging from 0.25 mcg to 2.0 mcg so as to maintain the concentration of calcium and phosphate at the upper normal range as in Study I.

The treatment group which included 13 patients.

These patients were administered a combination of 24,25(OH) 2

D

3 at 20 mcg/d and 1,25(OH) 2

D

3 at daily doses similiar to those administered to patients in control S group B.

The study showed the following results: I The combined treatment induced a less marked increment in serum calcium than that observed in the control group i 30 treated with 1,25(OH) 2

D

3 alone.

However, the decrement in alkaline phosphatase activity in the treatment group appeared to be less marked than in patients given 1,25(OH) 2

D

3 alone.

5996S:ND 22 5996S:ND 9 The following conclusions may be drawn from the study: The addition of 24,25(OH) 2

D

3 with 1,25(OH)2D 3 for the treatment of renal bone disease does not aggravate hypercalcemia, nor does it impair the apparent effectiveness of 1,25(OH) 2

D

3 in the treatment of hyperparathyroid bone disease.

The combination of 1,25(OH) 2

D

3 and 24,25(OH) 2

D

3 hp3 td disease in the treatment of hyperparthyroid bone disease is of value in those patients who had previously been shown to be refractory to treatment with 1,25(OH)2D 3 alone.

S ii i 5996S:ND 23

Claims

1. A method for the treatment of osteitis fibrosa cystica disease in human patients comprising administering to said patients a combination of 3 to 5 micrograms of 24,25-dihydroxycholecalciferol and 0.25 to 1.0 micrograms of 1-alpha-hydroxycholecalciferol or of 1,25-dihydroxy- cholecalciferol or 0.5 to 1.0 milligrams of dihydrotachysterol (DHT 2

2. A method according to claim 1, wherein said combination comprises 5 micrograms of 24,25-dihydroxy- cholecalciferol and 0.25 to 1.0 micrograms of 1-alpha- hydroxycholecalciferol or of 1,25-dihydroxycholecalciferol 0, 1 or 0.5 to 1.0 milligrams of DHT 2 So 5

3. A method according to claim 1, wherein said ooo: combination comprises a medicament in dosage unit form, said S°o combination in a mixture with an inert pharmaceutical I 000 S 0 carrier, said mixture in the form of tablets, pills, a o0 0 dragees, capsules, ampoules or suppositories.

4. A method of claim 3, wherein the mixture is in the form of a soft gelatin capsule.

A method according to claim 1, wherein the 0 o combination further comprises a stabilizing agent comprising Vitamin E, citric acid, propyl gallate and arachis oil. 25

6. A method according to claim 1, wherein the o:a combination is administered per day as follows: 6-40 micrograms per day of 24,25-dihydroxycholecalciferol in combination with a compound selected from the group consisting of l-alpha-hydroxycholecalciferol and 1,25- 0 30 dihydroxycholecalciferol, in an amount per day which will 0 0 o* "maintain the patient's calcium serum concentration at 10 to 11 mg/dl and phosphate serum concentration at 5.5-6.0 mg/dl.

7. A method according to claim 6, in which the numerical value of the serum calcium concentration multiplied by the numerical value of the serum phosphate concentration results in a numerical product of 60 to 7 9 96S/NL 24 means of ampoules containing the desired amounts of the 5996S:ND 12 1 i 1

8. A method according to claim 6, wherein dihydrotachysterol is administered with the 24,25-dihydroxycholecalciferol.

9. A method according to claim 6, wherein the 24,25-dihydroxycholecalciferol is administered in an amount of 10-40 mcg/d.

A method according to claim 6, wherein the 24,25-dihydroxycholecalciferol is administered in an amount of 10 mcg/d.

11. A method according to claim 1, wherein the combination is administered twice a day.

12. A method according to claim 1, wherein the composition further comprises parathyroid hormone (PTH).

13. A method according to claim 1, wherein the combination consists essentially of 24,25-dihydroxy- cholecalciferol and dihydrotachysterol.

14. A method according to claim 1, wherein the combination consists essentially of 24,25-dihydroxy- cholecalciferol and l-alpha-hydroxycholecalciferol. S 20

15. A medicament substantially as herein described wth i reference to any one of the Examples thereof. Dated this 15th day of August 1990 4C 425 TEVA PHARMACEUTICAL INDUSTRIES LTD. i By their Patent Attorney GRIFFITH HACK CO. 3 C 6S/NL 25